ISSN 2319-8885

Volume.08,
Jan-Dec-2019,
Pages:284-287
www.ijsetr.com

Investigation of Bioactive Compounds from The Bark of Smanea

Saman(Jaeq) Merr.(Thinbaw-Kokko)
YIN YIN THEIN1, KYAWT KYAWT KHAING2, KHIN SEIN MYINT3
1
Dept of Chemistry, Technological University, Magway, Myanmar, Email: yinyinthein.edu@gmail.com.
2
Dept of Chemistry, Technological University, Magway, Myanmar, Email: winterkk34@gmail.com.
3
Dept of Biotechnology, Technological University, Yamethin, Myanmar, Email: khinseinmyint.edu@gmail.com.

Abstract: In this paper, the bark of Samanea saman (Jacq)Merr. (Thinbaw-Kokko) was collected from Min-Hla Township,
Magway Division. Preliminary phytochemical tests on bark of Kokko revealed the presence alkaloid, terpene, steroid, reducing
sugar, saponin, phenolic compound, glycoside, tannin and polyphenol and the absence of flavonoid on treating with respective
reagents. In Vitro screening of antimicrobial activity was also carried out using the agar well diffusion method. Different extracts
such as n-hexane, CHCL3, acetone, EtOAc, EtOH and H2O extracts were used for this purpose. Among these extracts, CHCL3
extract possess the most potent antimicrobial activity with inhibition zone diameters of 20 ~ 28 mm. For isolation of bioactive
compounds, the bark of Kokko was extracted with 95% ethanol and then partitioned with EtOAc. Two bioactive organic
compounds (YYT-3 andYYT-4) were isolated. From the view of determination of the functional groups by using FT-IR
spectroscopic method, colour reaction test, (Alkaloid Test) and melting point measurement, one of the isolated compounds
(YYT-4) should be pithecolobine.

Keywords: Spectroscopic, Bark, Vitro, Presence Alkaloid, Antimicrobial.

I. BOTANICAL ASPECTS
Description
Botanical name - Samanea saman(Jacq) Merr.
Family - Mimosaceae
Myanmar name - Thinbow-Kokko
English name - Raintree

Figure2. The Tress of Thinbaw-Kokko.

II. CHARACTERISTIC FEATURE

Figure 1. The bark of Thinbaw-Kokko.
A large, tropical tree growing up to 180 feet tall with a
crown up to 240 feet proceed although they are smaller in
the sub-tropics. It is a beautiful shade tree. The leaves are
alternate bipinnate, while the flowers are small pinkish
green. The leaves are 2-4 pinnate with more or less elliptical
2-8 pinnate leaflets. Leaves are 20-24 cm long, evergreen,

Copyright @ 2019 IJSETR. All rights reserved.

 YIN YIN THEIN, KYAWT KYAWT KHAING, KHIN SEIN MYINT
fernlike, light green, dainty, shiny on the upper surface
underneath leaflets are spread out when it is sunny and
during the day. Flor*'ers are 6-10 mm in size borne on a 10-
13 cm stem. They are produced from spring to fall and add
beauty to an already attractive tree. Fruits are straight, shiny
when unripe and dark brown to black when ripe are 15-20
cm long. The fruits contain sticky brown pulp and are
relished as cattle fodder.
Part used - Bark
Medical uses- Malaria, chronic illness, fever, indigestion,
diarrhoeal diseases and injuries.
III. EXPERIMENTAL RESULT
A. Sample Collection
The bark of Thinbaw-Kokko was collected from Min-Hla
Township, Magway Division. The bark was cut into small
pieces and allowed to dry by air in the shade.
B. Percolation
The pieces of the bark of Kokko (400 g) were percolated
with 95% ethanol (1600ml) for two months. Then the
resulting ethanolic solution was filtered and the filtrate was
concentrated.
C. Phytochemical Screening
Phytochemical tests of the bark of Samanea saman (Jacq)
Merr. were done and the results were shown in Table(1). It
can be seen that the bark of Kokko contain alkaloid,
polyphenol, terpene, steroid, reducing Sugar, glycoside,
phenolic compound, saponin and tannin.
D. Test for Alilrotd
About (2g) of the bark of Kokko sample was boiled with
1% hydrochloric acid (10 mL) about 10 min, allowed to cool
and filtered. The filtrate was divided into two portions in two precipitate was observed indicating that reducing sugar may
test tubes. These portions were tested with Dragendroff’s
reagent and Wagner's reagent. The formation of orange ppt
and yellow ppt indicated the presence of alkaloids
respectively.
E. Test for Flavonoid
The sample about (2g) was dissolved in 10 mL of 95 %
ethanol, boiled about 10 min and then filtered and cool. Then
0.5 ml of concentrated HCl and a few milligram of (Mg)
turnings were added in this filtrate. No pink colour was
observed indicating the absence of flavonoid in Kokko.
F. Test for Polyphenol
The sample about (2g) was boiled with EtOH (10mL)
about 10 min and then filter and cool, 3 mL of 1 % FeCl3,
and Kr3[Fe(CN)6] solution was added to the filtrate where
greenish blue colour was observed. Therefore, polyphenol
was present in Thinbaw-Kokko.
G. Test for Terpene
The sample nearly (2g) was boiled with EIOH (10mL)
about l0 min and then filtered. To this filtrate, 1 mL of acetic
anhydride, I mL of chloroform and 2 mL of conc: H 2SO4
International Journal of Scientific Engineering and Technology Research
Volume.08, Jan-Dec-2019, Pages: 284-287
were added and shaken. The red color was observed, so it is
indicated the presence of terpene.
H. Test for Glycoside
The sample was boiled with distilled water (10 mL) about
10 min, allowed to cool and filtered. The filtrate was treated
with 10% lead acetate solution. The white precipitates was
observed, and so glycoside may be present.
I. Test for Sterol
The sample was heated with 10 mL CHCL3. The mixture
was filtered and filtrate was obtained. Then 2-3 drops of
concentration H2SO4 was added to the filtrate where green
colour was observed. Therefore, sterol was present in
Kokko.
J. Test for Saponin
The sample (2g) was boiled with distilled water (10 mL)
about 10 min and it was vigorously shaken for a few
minutes. The permanent foam was observed indicating that
saponin may be present.
K. Test for Tannin
The sample nearly (2g) was boiled with distilled water
(10mL) about 10 min and then filtered. The filtrate was
treated with 0.5 mL of 2% NaCl and then the clear solution
from filtrate was added with 0. 5 ml l% FeCl3. The
appearance of yellowish brown colour showed the presence
of tannins.
L. Test for Reducing Sugar
The sample about (2g) was boiled with distilled water
(10mL) about 10 min and then filtered. The filtrate was
boiled with Benedict's solution. The formation of brick red
be present in Kokko.
M. Test for Phenolic Compound
The sample was heated with 10 mL EtOH. The mixture
was filtered and filtrate was obtained. Then 5-10 drops of
l0% FeCl3, solution was added to the filtrate where green
colour was observed. Therefore, phenolic compound was
present in Thinbaw-Kokko.
Table 1. Results of Phytochemical Tests on bark of
Thinbaw-Kokko
 Investigation of Bioactive Compounds from The Bark of Smanea Saman(Jaeq) Merr.(Thinbaw-Kokko)
IV. ANTIMICROBIAL ACTIVITY OF THE BARK OF room temperature. The residue was dissolved in 150 mL of
THINBAW-KOKKO ethyl acetate. Ethyl acetate extract solution was filtered and
Various extracts of plant sample were sent to DCPT evaporated to give (1.8 g) of crude extract.
(Development centre for pharmaceutical technology), Insein,
Yangon. Antimicrobial activity of extracts of bark of Kokko B. Column Separation of Crude Extract from the bark of
was tested. Table2 represents the results of the antimicrobial Thinbaw-Kokko
test relevant to six types of organisms. The EtOAc crude extract (1.8 g) was chromatographed on
silica gel (70 -230 mesh), eluting with n-hexane and ethyl
Table2. Results of Antimicrob i al Activity of the B ark of cetate with various ratios from non-polar to polar to produce
Thinbaw-Kokko (174) fractions. Each fraction was checked by TLC. When
the same Rf value fractions were combined, thirteen
combined fractions were obtained. The fraction (8) was the
main fraction and was checked again by TLC to give
compound YYT-4 (Rf =0.40) in n-hexane: EtOAc (4: 1 V/V)
solvent system. The percent yield was found to be 2.l%
based upon the crude extract. Other compound was found to
be fraction (6) and designed as YYI-3 (Rf = 0.40), 3.3% also
in the above solvent system.

The air dried powder (400 g)

1. Percolated with EtOH (1600 ml) for two months
2. Filtered and evaporated
Residue
According to this table, CHCL extract gives high activity Was extracted with EtOAc and concentrated
on dl tested organisms. The extract of ethyl acetate responds
medium to high activity on all selected organisms. Acetone EtOAc extract (1.8 g)
extract gives medium activity on six selected organisms. The Separated by Column Chromatrgraphy n-hexane:
extract of ethanol responds low to medium activity on six EtOAc (various rations)
selected organisms. (174) total fractions

Checked by TLC
The same Rf value fractions were combined (13)

Combined factions

6 8

Needle shape crystal (YYT-3) Major fraction

(0.06g)(3.3%) amorphous form (YYT-4)
Rf =0.45 (0.04g)(2.1%)
(n-hexane: EtOAc 4:1 V/V) Rf =0.40
(n-hexane: EtOAc 4:1 V/V)
Figure4. Extraction and isolation of compounds yyT-4
and yyT:3 from Kokko.

1. n-hexane, 2. CHCI3, 3. Acetone 4. EtOAc, 5. EIOH, 6.H2O

Figure 3. Inhibition zones of various extracts of
Thinbaw-Kollko on six types of organisms.

V. EXTRACTION AND ISOLATION OF THE MAIN

CONSTITUENTS FROM THE BARK OF THINBAW-KOKKO
A. Preparation of Crude Extracts from Thinbaw-Kokko
Air-dried powder of the bark of Kokko (400 g) was
percolated with 95% ethyl alcohol (1600 mL) for two Figure5. Separation of the crude extract by column
months. Percolated solution was filtered and evaporated at chromatography

International Journal of Scientific Engineering and Technology Research

Volume.08, Jan-Dec-2019, Pages: 280-283
 YIN YIN THEIN, KYAWT KYAWT KHAING, KHIN SEIN MYINT
VI. RESULTS AND DISCUSSION
The FT-IR spectrum of compounds was measured at the
department of Chemistry University of Mandalay and they
were described in figure (5.1) and figure (5.3) respectively. [3]Harborne, J.8., (1993), "Methods in Plant Biochemistry"
[2] Hamerslag, F.E., (1950), "The Technology and
Chemistry ofAlkaloids", D. Van Nostrand Cornpany, Inc.,
New York, 8 1 -95
Academic Press, London, VIII,397
[4] Harborne, J.B., (1993), "Phytochemical Dictionary:
AHandbookof Bioactive Compounds from Plants," Taylor&
Francis Ltd., London, Washington DC
[5] Manjuneath, B.I., (1952) "TheWealth oflndia, A
Dictionary oflndian Raw Materials and Industrial Products",
lll, 17 5-177
[6] Prasad, R.N.etal, (2008), "Preliminary Phytochemical
Screening and Antimicrobial Activify of Samoneo
soma4"Journal of Medicinal Plants Research,2(10), 268-27 0
[7] Rastogi, R.P. and Mehrotra,8.N., (1993), "Compendium
of Indian Medicinal Plants",Central Drug Research Institute,
Lucknow andNational Institute of “Science Communication.
New Delhi.II.607
[8] Roberts, M.F.and Wink, M., (1998), "Alkaloids:
Biochemistry, Ecology and Medicinal Applications,"
Plenum Press, New York, 1-6
[9]Stephen, K.S., (1965), "”Medicinal Plant Alkaloids” 2nd
Edition, University of Toronto Press, 54-66
[10] Vogel, A" L., (1956), “A Textbook of Practical Organic
Chemistryr”, 5th Edition, Language Book Society and
Longman Group Ltd., London, 454
[11] http://www.en.wikipedia.org/wiki/Alkaloid
[12] http://www.people.vcu.edu/-asneden/alkaloids.htm.

Figure.6. The FT-IR spectrum of compound.

In this experimental work, the bark of Thinbaw-Kokko

was collected from Min-Hla Township, Magway Division.
The antimicrobial activity of crude extracts of the bark of
Kokko in various solvent systems was tested by agar well
diffusion method on six selected organisms. Among them,
CHCI3 extract gave high activity on all tested six organisms.
Furthermore, the presence of alkaloid, terpene, steroid,
reducing sugar, saponin, phenolic compound, glycoside,
tannin and polyphenol and absence of flavonoid could be
observed by phytochemical screening of the crude extracts
of the sample. One alkaloid compound (M.pt 68 - 70C,
2.1%yield) was isolated from EtOAc extract by modern
separation techniques such as Thin layer and Column
Chromatography. Its Rf value is found to be 0.40 inn-hexane:
EtOAc (a:1 VA/) solvent system. From functional groups
determination by FT:IR spectroscopy, colour reaction
(alkaloid test) and melting point value, the isolated
compound YYL4 could be identified as pithecolohine, one
of the constituents of Kokko.

VII. REFERENCES
[1] Buchanan, B., Guissem, W. and Jones. R., (2000)
"Biochemistry & Molecular Biology Plants", American
Socieqv of Plant Physiologists, John Wiley and Sons.
NewYork.

International Journal of Scientific Engineering and Technology Research

Volume.08, Jan-Dec-2019, Pages: 284-287